Abstract

In microorganisms, Ion Torrent sequencing technology has been proved to be useful in whole-genome sequencing of bacterial genomes (5 Mbp). In our study, for the first time we used this technology to perform a resequencing approach in a whole fungal genome (36 Mbp), a non-ochratoxin A producing strain of Aspergillus carbonarius. Ochratoxin A (OTA) is a potent nephrotoxin which is found mainly in cereals and their products, but it also occurs in a variety of common foods and beverages. Due to the fact that this strain does not produce OTA, we focused some of the bioinformatics analyses in genes involved in OTA biosynthesis, using a reference genome of an OTA producing strain of the same species. This study revealed that in the atoxigenic strain there is a high accumulation of nonsense and missense mutations in several genes. Importantly, a two fold increase in gene mutation ratio was observed in PKS and NRPS encoding genes which are suggested to be involved in OTA biosynthesis.

@article{osti_1215424,
title = {Rapid genome resequencing of an atoxigenic strain of Aspergillus carbonarius},
author = {Cabañes, F. Javier and Sanseverino, Walter and Castellá, Gemma and Bragulat, M. Rosa and Cigliano, Riccardo Aiese and Sánchez, Armand},
abstractNote = {In microorganisms, Ion Torrent sequencing technology has been proved to be useful in whole-genome sequencing of bacterial genomes (5 Mbp). In our study, for the first time we used this technology to perform a resequencing approach in a whole fungal genome (36 Mbp), a non-ochratoxin A producing strain of Aspergillus carbonarius. Ochratoxin A (OTA) is a potent nephrotoxin which is found mainly in cereals and their products, but it also occurs in a variety of common foods and beverages. Due to the fact that this strain does not produce OTA, we focused some of the bioinformatics analyses in genes involved in OTA biosynthesis, using a reference genome of an OTA producing strain of the same species. This study revealed that in the atoxigenic strain there is a high accumulation of nonsense and missense mutations in several genes. Importantly, a two fold increase in gene mutation ratio was observed in PKS and NRPS encoding genes which are suggested to be involved in OTA biosynthesis.},
doi = {10.1038/srep09086},
journal = {Scientific Reports},
number = ,
volume = 5,
place = {United States},
year = 2015,
month = 3
}

Plant biomass, once reduced to its composite sugars, can be converted to fuel substitutes. One means of overcoming the recalcitrance of lignocellulose is pretreatment followed by enzymatic hydrolysis. However, currently available commercial enzyme cocktails are inhibited in the presence of residual pretreatment chemicals. Recent studies have identified a number of cellulolytic enzymes from bacteria that are tolerant to pretreatment chemicals such as ionic liquids. The challenge now is generation of these enzymes in copious amounts, an arena where fungal organisms such as Aspergillus niger have proven efficient. Fungal host strains still need to be engineered to increase production titers ofmore » heterologous protein over native enzymes, which has been a difficult task. Here, we developed a forward genetics screen coupled with whole-genome resequencing to identify specific lesions responsible for a protein hyper-production phenotype in A. niger. As a result, this strategy successfully identified novel targets, including a low-affinity glucose transporter, MstC, whose deletion significantly improved secretion of recombinant proteins driven by a glucoamylase promoter.« less

Ochratoxin A (OTA), a mycotoxin produced by Aspergillus and Penicillium species, is composed of a dihydroisocoumarin ring linked to phenylalanine and its biosynthetic pathway has not yet been completely elucidated. Most of the knowledge regarding the genetic and enzymatic aspects of OTA biosynthesis has been obtained in Penicillium species. In Aspergillus species only pks genes involved in the initial steps of the pathway have been partially characterized. In our study, the inactivation of a gene encoding a nonribosomal peptide synthetase in OTA producing A. carbonarius ITEM 5010 has removed the ability of the fungus to produce OTA. This is themore » first report on the involvement of an nrps gene product in OTA biosynthetic pathway in Aspergillus species. The absence of OTA and ochratoxin α-the isocoumaric derivative of OTA, and the concomitant increase of ochratoxin β- the dechloro analog of ochratoxin α- were observed in the liquid culture of transformed strain. The data provide the first evidence that the enzymatic step adding phenylalanine to polyketide dihydroisocoumarin precedes the chlorination step to form OTA in A. carbonarius, and that ochratoxin α is a product of hydrolysis of OTA, giving an interesting new insight in the biosynthetic pathway of the toxin.« less

Ochratoxin A (OTA) is a potent mycotoxin produced by Aspergillus and Penicillium species and is a common contaminant of a wide variety of food commodities, with Aspergillus carbonarius being the main producer of OTA contamination in grapes and wine. The molecular structure of OTA is composed of a dihydroisocoumarin ring linked to phenylalanine and, as shown in different producing fungal species, a polyketide synthase (PKS) is a component of the OTA biosynthetic pathway. Similar to observations in other filamentous ascomycetes, the genome sequence of A. carbonarius contains a large number of genes predicted to encode PKSs. In this work amore » pks gene identified within the putative OTA cluster of A. carbonarius, designated as AcOTApks, was inactivated and the resulting mutant strain was unable to produce OTA, confirming the role of AcOTApks in this biosynthetic pathway. AcOTApks protein is characteristic of the highly reduced (HR)-PKS family, and also contains a putative methyltransferase domain likely responsible for the addition of the methyl group to the OTA polyketide structure. AcOTApks is different from the ACpks protein that we previously described which showed an expression profile compatible with OTA production. We performed phylogenetic analyses of the β-ketosynthase and acyl-transferase domains of the OTA PKSs which had been identified and characterized in different OTA producing fungal species. The phylogenetic results were similar for both the two domains analyzed and showed that OTA PKS of A. carbonarius, Aspergillus niger, and Aspergillus ochraceus clustered in a monophyletic group with 100% bootstrap support suggesting a common origin, while the other OTA PKSs analyzed were phylogenetically distant. A qRT-PCR assay monitored AcOTApks expression during fungal growth and concomitant production of OTA by A. carbonarius in synthetic grape medium. A clear correlation between the expression profile of AcOTApks and kinetics of OTA production was observed with AcOTApks which reached its maximum level of transcription before OTA accumulation in mycelium reached its highest level, confirming the fact that gene transcription always precedes phenotypic production.« less